Somatic hypermutation and class switching in autoimmunity

描述（由申请人提供）：我们希望了解系统性自身免疫（特别是系统性狼疮）中抗体体细胞超突变（SHM）和类别转换DNA重组（CSR）的分子机制。像微生物病原体的抗体一样，大多数致病性自身抗体是体细胞超突变和类别转换的。SHM主要在免疫球蛋白（IG）重链和轻链V（D）J区引入点突变，从而为通过（自身）抗原选择更高亲和力（自身）抗体突变体提供底物。CSR替换表达的IgH恒定（CH）区，例如，C5，与下游C3、C1或C5，从而赋予（自身）抗体新的生物效应子功能。SHM和CSR都需要两个连续的阶段：（i）由激活诱导的胞苷脱氨酶（AID）介导的DNA损伤的产生，以及（ii）通过基本位点旁路、碱基切除修复（BER）或错配修复（MMR）机制进行的DNA损伤修复，导致错配（SHM）或双链DNA断裂（DSB）的出现及其解决（CSR）。我们认为，在系统性狼疮，SHM和CSR是失调，由于上调AID，因此，增加DNA损伤，并改变DNA修复。我们认为，如我们所示，关键调节SHM和CSR的遗传学保守的同源结构域蛋白HoxC 4通过直接结合到我们在AID启动子中鉴定的保守的HoxC 4/Oct-binding 5 '-ATTTGAAT-3'位点来诱导AID表达。我们还认为，雌激素通过触发雌激素受体（ER）与我们最近在HoxC 4启动子中发现的两个保守的雌激素反应元件（ERE）结合，大大增强了CD 154：CD 40诱导的HoxC 4表达，从而进一步增强了AID表达，并导致狼疮中SHM/CSR失调。我们还认为，translesion DNA synthesis（TLS）聚合酶（pol）8和pol 7在（自身）抗体的SHM中是至关重要的，通过修复DNA损伤后，由泛素化的PCNA，介导的“聚合酶开关”从高保真复制型DNA聚合酶的易错TLS聚合酶招募。最后，我们假设HoxC 4、AID和TLS聚合酶的表达在狼疮易感小鼠中失调，HoxC 4、Ung或TLS pol 7的消融导致SHM/CSR降低，因此导致高亲和力和同种型转换的自身抗体水平降低和免疫病理学延迟。为了验证我们的假设，我们将：（i）确定HoxC 4介导的AID启动子激活和雌激素增强HoxC 4以及因此增强AID表达的分子机制;（ii）阐明TLS pol 8和pol 7的作用及其在SHM中被泛素化PCNA募集的作用;最后，（iii）分析SHM/CSR失调和HoxC 4、Ung org pol 7缺陷对狼疮易感小鼠中高度突变和类别转换自身抗体产生的影响。这些实验将使用复杂的生物化学方法和我们的新KO HoxC 4-/-、pol 8-/-、双KO pol 8-/-Ung-/-和pol 7-/-Ung-/-以及突变型Aicdatm 1（Pmut）和pol 7 mut小鼠，以及HoxC 4、Ung或pol 7缺陷的狼疮易感MRL/Faslpr/lpr小鼠。

DESCRIPTION (provided by applicant): We want to understand the molecular mechanisms that underlie antibody somatic hypermutation (SHM) and class switch DNA recombination (CSR) in systemic autoimmunity, particularly systemic lupus. Like antibodies to microbial pathogens, most pathogenic autoantibodies are somatically hypermutated and class-switched. SHM introduces mainly point-mutations in immunoglobulin (Ig) heavy and light chain V(D)J regions, thereby providing the substrate for selection by (self) antigen of higher affinity (auto)antibody mutants. CSR replaces the expressed IgH constant (CH) region, e.g., C5, with downstream C3, C1 or C5, thereby endowing (auto)antibodies with new biological effector functions. Both SHM and CSR entail two sequential stages: (i) generation of DNA lesions, as mediated by activation-induced cytidine-deaminase (AID), and (ii) DNA lesion repair by the basic site bypass, base-excision repair (BER) or mismatch repair (MMR) machineries, leading to the emergence of mismatches (SHM) or double-strand DNA breaks (DSBs) and their resolution (CSR). We argue here that in systemic lupus, SHM and CSR are dysregulated, as a result of upregulated AID and, therefore, increased DNA lesions, and altered DNA repair. We contend that the phylogenetically conserved homeodomain protein HoxC4, which, as we have shown, critically regulates SHM and CSR, induces AID expression by directly binding to a conserved HoxC4/Oct-binding 5'-ATTTGAAT-3' site we have identified in the AID promoter. We also contend that estrogen greatly enhances CD154:CD40-induced HoxC4 expression by triggering the binding of estrogen receptors (ERs) to two conserved estrogen responsive elements (EREs) we have recently identified in the HoxC4 promoter, thereby further enhancing AID expression and contributing to SHM/CSR dysregulation in lupus. We also argue that the translesion DNA synthesis (TLS) polymerase (pol) 8 and pol7 are critical in SHM of (auto)antibodies, by repairing DNA lesions after being recruited by ubiquitinated PCNA, which mediates the "polymerase switch" from high-fidelity replicative DNA polymerases to error-prone TLS polymerases. Finally, we hypothesize that expression of HoxC4, AID and TLS polymerases is dysregulated in lupus-prone mice and ablation of HoxC4, Ung or TLS pol7 leads to decreased SHM/CSR and, therefore, decreased level of high-affinity and isotype-switched autoantibodies and delayed immunopathology. To test our hypotheses, we will: (i) define the molecular mechanisms of HoxC4-mediated AID promoter activation and the enhancement of HoxC4 and, therefore, AID expression by estrogen; (ii) address the role of TLS pol8 and pol7 and their recruitment by ubiquitinated PCNA in SHM; and, finally, (iii) analyze the SHM/CSR dysregulation and the impact of deficiency of HoxC4, Ung org pol7 on the generation of hypermutated and class-switched autoantibodies in lupus-prone mice. These experiments will use sophisticated biochemical methods and our new KO HoxC4-/-, pol8-/-, double KO pol8-/-Ung-/- and pol7-/-Ung-/- and mutant Aicdatm1(Pmut) and pol7mut mice, and lupus-prone MRL/Faslpr/lpr mice deficient in HoxC4, Ung or pol7.